Branched chain fatty acids (BCFA) are mostly saturated fatty acids (SFA) with one or more methyl branches on the carbon chain. BCFA are synthesized mainly by the skin and have long been known to be a major component of vernix caseosa (10-20% dry weight) (Nicolaides et al., “Skin Lipids. 3. Fatty Chains in Skin Lipids. The Use of Vernix Caseosa to Differentiate between Endogenous and Exogenous Components in Human Skin Surface Lipid,” J Am Oil Chem Soc 42:702-707 (1965)). Among terrestrial animals, vernix is unique to humans, and is not found in other land mammals, including other primates (Pickens et al., “Characterization of Vernix Caseosa: Water Content, Morphology, and Elemental Analysis,” J Invest Dermatol 115:875-881 (2000)). Vernix is made of sebum and fetal corneocytes (Nicolaides et al., “Skin Lipids. 3. Fatty Chains in Skin Lipids. The Use of Vernix Caseosa to Differentiate between Endogenous and Exogenous Components in Human Skin Surface Lipid,” J Am Oil Chem Soc 42:702-707 (1965) and Narendran et al., “Interaction between Pulmonary Surfactant and Vernix: A Potential Mechanism for Induction of Amniotic Fluid Turbidity,” Pediatr Res 48:120-124 (2000)) and is produced by fetal skin starting at 24 weeks gestational age and continuing until term birth (Moore et al., “Fetal Cocaine Exposure: Analysis of Vernix Caseosa,” J Anal Toxicol 20:509-511 (1996)). During the third trimester vernix sloughs off as particulates that become suspended in amniotic fluid (Narendran et al., “Interaction between Pulmonary Surfactant and Vernix: A Potential Mechanism for Induction of Amniotic Fluid Turbidity,” Pediatr Res 48:120-124 (2000) and Yoshio et al., “Antimicrobial Polypeptides of Human Vernix Caseosa and Amniotic Fluid: Implications for Newborn Innate Defense,” Pediatr Res 53:211-216 (2003)), possibly aided by lung surfactant phospholipids that also enter the amniotic fluid. The fetus normally swallows amniotic fluid in amounts approaching 500 ml at the end of gestation (Miettinen et al., “Gas-liquid Chromatographic and Mass Spectrometric Studies on Sterols in Vernix Caseosa, Amniotic Fluid and Meconium,” Acta Chem Scand 22:2603-2612 (1968) and Sherman et al., “Fetal Swallowing: Correlation of Electromyography and Esophageal Fluid Flow,” Am J Physiol 258:R1386-1394 (1990)) and with it vernix. Thus, the late term fetal gut is normally exposed to vernix and its BCFA, increasingly so as parturition approaches.
Vernix dry matter is composed of approximately equal amounts of protein and lipids (Pickens et al., “Characterization of Vernix Caseosa: Water Content, Morphology, and Elemental Analysis,” J Invest Dermatol 115:875-881 (2000) and Hoeger et al., “Epidermal Barrier Lipids in Human Vernix Caseosa: Corresponding Ceramide Pattern in Vernix and Fetal Skin,” Br J Dermatol 146:194-201 (2002)). Lipid fractions in vernix have been comprehensively characterized (Nicolaides et al., “The Fatty Acids of Wax Esters and Sterol Esters from Vernix Caseosa and from Human Skin Surface Lipid,” Lipids 7:506-517 (1972); Rissmann et al., “New Insights into Ultrastructure, Lipid Composition and Organization of Vernix Caseosa,” J Invest Dermatol 126:1823-1833 (2006) and Kaerkkaeinen et al., “Lipids of Vernix Caseosa,” J Invest Dermatol 44:333-338 (1965)) and shown to be 25-30% sterol esters (SE), 18-36% triglycerides (TAG), 12-16% wax esters (WE), 9% squalene, 5% ceramides, and low levels of non-esterified fatty acid (NEFA) fraction was also detected by some (Rissmann et al., “New Insights into Ultrastructure, Lipid Composition and Organization of Vernix Caseosa,” J Invest Dermatol 126:1823-1833 (2006) and Tollin et al., “Vernix Caseosa as a Multi-component Defence System Based on Polypeptides, Lipids and Their Interactions,” Cell Mol Life Sci 62:2390-2399 (2005)) but not by others (Nazzaro-Porro et al., “Effects of Aging on Fatty Acids in Skin Surface Lipids,” J Invest Dermatol 73:112-117 (1979)). BCFA are found in all acyl-carrying lipid classes, WE (16-53%) and SE (27-62%) (Nicolaides et al., “The Fatty Acids of Wax Esters and Sterol Esters from Vernix Caseosa and from Human Skin Surface Lipid,” Lipids 7:506-517 (1972); Rissmann et al., “New Insights into Ultrastructure, Lipid Composition and Organization of Vernix Caseosa,” J Invest Dermatol 126:1823-1833 (2006); Kaerkkaeinen et al., “Lipids of Vernix Caseosa,” J Invest Dermatol 44:333-338 (1965) and Nazzaro-Porro et al., “Effects of Aging on Fatty Acids in Skin Surface Lipids,” J Invest Dermatol 73:112-117 (1979)), as well as in the TAG (18-21%) and NEFA (21%) fractions (Rissmann et al., “New Insights into Ultrastructure, Lipid Composition and Organization of Vernix Caseosa,” J Invest Dermatol 126:1823-1833 (2006)).
Apart from skin (Nicolaides et al., “Skin Lipids. 3. Fatty Chains in Skin Lipids. The Use of Vernix Caseosa to Differentiate between Endogenous and Exogenous Components in Human Skin Surface Lipid,” J Am Oil Chem Soc 42:702-707 (1965); Nicolaides et al., “The Fatty Acids of Wax Esters and Sterol Esters from Vernix Caseosa and from Human Skin Surface Lipid,” Lipids 7:506-517 (1972) and Nicolaides et al., “Skin Lipids: Their Biochemical Uniqueness,” Science 186:19-26 (1974)), BCFA are at very low levels in internal tissue (Nicolaides et al., “Skin Lipids: Their Biochemical Uniqueness,” Science 186:19-26 (1974)), but are also found in human milk (Jensen et al., “Handbook of Milk Composition,” Academic Press Inc., San Diego, (1995); Egge et al., “Minor Constituents of Human Milk. IV. Analysis of the Branched Chain Fatty Acids,” Chem Phys Lipids 8:42-55 (1972) and Gibson et al., “Fatty Acid Composition of Human Colostrum and Mature Breast Milk,” Am J Clin Nutr 34:252-257 (1981)) at concentrations as high as 1.5% w/w of total fatty acids (FA). This level is comparable to and in some cases greater than that of docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (ARA, 20:4n-6) in the same milk. For instance, a 1981 publication reported the concentration of anteiso 17:0 in Australian women's colostrum to be 0.45% w/w of total FA, exceeding the concentrations of DHA (0.32% w/w) and ARA (0.4% w/w) (Gibson et al., “Fatty Acid Composition of Human Colostrum and Mature Breast Milk,” Am J Clin Nutr 34:252-257 (1981)).
Meconium, the newborn's first fecal pass, first appears in the fetal GI tract at around 12 weeks of gestational age, and is normally passed after birth (Ahanya et al., “Meconium Passage In Utero: Mechanisms, Consequences, and Management,” Obstet Gynecol Surv 60:45-56 (2005); Gareri et al., “Drugs of Abuse Testing in Meconium,” Clin Chim Acta 366:101-111 (2006); and Ostrea et al., “Fatty Acid Ethyl Esters in Meconium: Are They Biomarkers of Fetal Alcohol Exposure and Effect?” Alcohol Clin Exp Res 30:1152-1159 (2006)). It consists of amniotic fluid residue, skin and gastrointestinal (GI) epithelial cells, GI secretions and enzymes, lipids, sugars, proteins, cholesterol, sterols, bile acid and salts (Ahanya et al., “Meconium Passage In Utero: Mechanisms, Consequences, and Management,” Obstet Gynecol Surv 60:45-56 (2005); Gareri et al., “Drugs of Abuse Testing in Meconium,” Clin Chim Acta 366:101-111 (2006); Buchanan et al., “Chemical Comparison of Normal Meconium and Meconium from a Patient with Meconium Ileus,” Pediatrics 9:304-310 (1952); and Righetti et al., “Proton Nuclear Magnetic Resonance Analysis of Meconium Composition in Newborns,” J Pediatr Gastroenterol Nutr 36:498-501 (2003)). Meconium contains 12% dry weight lipid (Buchanan et al., “Chemical Comparison of Normal Meconium and Meconium from a Patient with Meconium Ileus,” Pediatrics 9:304-310 (1952)), and there is only one unconfirmed study reporting BCFA in meconium (Terasaka et al., “Free Fatty Acids of Human Meconium,” Biol Neonate 50:16-20 (1986)). There are no studies linking BCFA composition of vernix and meconium in the same infants.
It was hypothesized that vernix BCFA of term newborns would survive the alimentary canal and be found in meconium. The test of this hypothesis led to characterizing the relative BCFA profiles of vernix and meconium to establish the degree to which the profile is altered by the sterile fetal gut in utero.
The present invention is directed to overcoming the deficiencies in the art.